26 research outputs found

    Characterization of HA and NA-containing VLPs produced in suspension cultures of HEK 293 cells

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    Virus like particles (VLPs) can be formulated into promising vaccines to prevent influenza infection. In addition of having a structure and composition that mimic the wild type virus, VLPs are safe since they are devoid of viral genes and consequently are not infectious. One approach to scale up the manufacturing of VLPs is to produce them in a serum-free suspension culture using a stable mammalian cell line. Importantly, with VLPs synthetized by mammalian cells, the post-translational modifications of the surface antigens should be similar to the wild type virus, and therefore should trigger a potent and specific immune response for the pathogen. As a proof of concept, we first established a cell line that was stably expressing hemagglutinin (HA) and neuraminidase (NA) proteins of influenza (subtype H1N1) using our patented cGMP human embryonic kidney (HEK293) cell line (clone 293SF-3F6). Transcription of the genes for these two glycoproteins was regulated by the inducible cumate transcription gene-switch. Next, to establish our capability to produce VLPs, we compared the formation of VLPs using these cells after forced expression of two scaffold proteins: Gag from the human immunodeficiency virus and M1 protein from influenza A (H1N1). In addition, monitoring of the VLPs was facilitated by fusing the Gag protein to the green fluorescent protein (GFP). VLP production was therefore initiated by transient transfection of plasmid encoding Gag or M1 and by addition of cumate to the culture medium. The VLPs secreted in the culture medium were recovered by ultracentrifugation on a sucrose cushion. The presence of HA an NA within the VLP fraction was demonstrated by western blot and quantified by dot blot. Interestingly, VLPs were produced more efficiently in the presence of Gag, indicating that Gag is a better scaffolding protein than M1 in this context. Under the electron microscope, the Gag-VLPs appeared as vesicles of 100 to 150 nm of diameter, containing a denser internal proteinous ring, which is a typical morphology for VLPs produced through Gag expression. The production of Gag-VLPs was also validated in a 3-L stirred tank bioreactor in serum-free medium. The immunogenicity of the VLPs is currently under investigation in a murine model for influenza. In conclusion, VLPs containing HA and NA can be manufactured in serum free suspension culture of HEK293 cells through forced expression of Gag. The efficacy of these VLPs for vaccination remains to be demonstrated

    Scalable lentiviral vector production using stable producer cell lines in perfusion mode

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    Lentiviral vectors (LVs) are becoming an important tool in gene and cell therapy and are being utilized in several clinical studies for rare and more frequent genetic and acquired diseases, as well as in cancer therapies. However, two major challenges need to be overcome in order to generate enough material to treat patients: First, current production platforms result in low titers (stable producer cell lines from adherent cell lines) or are not amenable to large scale production (LV produced by transfection). Next, LVs are known to have a low temperature stability. To address these two challenges, the National Research Council Canada has developed packaging cell lines and stable producer cell lines for the production of LVs which can grow in suspension in serum-free media and produce LV in the 106 TU/ml range without optimization. Furthermore, productions are performed in perfusion mode in order to operate at high cell densities and address the low LV stability. Please click Additional Files below to see the full abstrac

    Scalable lentiviral vector production using stable producer cell lines in perfusion mode

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    Lentiviral vectors (LVs) are becoming an important tool in gene and cell therapy and are being utilized in several clinical studies against genetic and acquired diseases, as well as in cancer therapies. To address the challenges linked to the generation of preclinical and clinical supply, the National Research Council Canada has developed packaging cell lines and stable producer cell lines for the production of LVs which can grow in suspension in serum-free media and produce LV in the 106 TU/ml range without optimization. We focus on the development of perfusion processes to both intensify the process and harvest produced LV rapidly

    Pan-HA antibodies for influenza detection and quantification

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    The influenza virus imposes a heavy burden for society in terms of health and economy. Influenza is an elusive enveloped virus due to antigenic shift and drift of two surface proteins: neuraminidase (NA) and hemagglutinin (HA). As a result, new strains emerge every year which require seasonal vaccination for protection. Furthermore, large vaccine quantities are urgently needed in case of pandemics. Theoretically, vaccines against a new strain can be manufactured in as little as three weeks with certain platforms and technologies. However, vaccine quantification and release are still relying on the use of the Single Radial Immunodiffusion (SRID) assay using a strain-specific antibody to calculate HA concentration. This is a major limitation because it can take up to three months to generate the reagents necessary to run the SRID assay, including the strain-specific antibody. Hence, one of the major hurdles in the process of influenza vaccine production is the quantification of HA which is critical to establish proper dosing. To circumvent the need for strain-specific antibodies, we have produced two monoclonal antibodies (F211-11H12-3 and F211-10A9-2) against a highly conserved peptide sequence found within the HA molecule (1). Multiple strains belonging to 13 different influenza A subtypes, as well as 6 strains belonging to B lineages were detected by Western blot and dot blot. Overall, mAb F211-11H12-3 recognizes preferentially influenza A subtype 1, while the mAb F211-10A9-2 has a higher affinity for influenza A subtype 2. Therefore, all strains tested could be detected when both mAb are combined and used as a cocktail. Next, we performed quantitative dot blots by generating a standard curve ranging from 160ng/ml to 20µg/ml HA. This method is simple, easy to implement and highly reproducible. In-process samples as well as purified material can be quantified by dot blot after denaturation with urea. Even though the SRID is the only assay approved by regulatory agencies, quantitative dot blots can be used during manufacturing to optimize and monitor the production process. Finally, ELISA is widely used for quantification and preliminary data demonstrates that samples can be quantified with the pan-HA mAbs. In conclusion, a pan-HA antibody cocktail was generated against a highly conserved peptide sequence of influenza. Viruses produced in eggs and mammalian cells from 40 different strains were detected by Western blot. Reproducible quantification was achieved by dot blot using the two mAbs and an appropriate calibrating standard. The combination of pan-HA antibodies with an immunoassay such as the dot blot assay could accelerate process development and help establish new generation quantification methods for influenza. As the field is looking for flexible and versatile solutions to shift away from the SRID assay and strain-specific antibodies, the development of broad-spectrum antibodies offers a long-awaited alternative. 1) Chun et al, Universal antibodies and their applications to the quantitative determination of virtually all subtypes of the influenza A viral hemagglutinins, Vaccine (26), pp 6068-6076, 2008

    Towards continuous bioprocessing of lentiviral vectors

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    Lentiviral vectors (LV) represent a key tool for cell and gene therapy applications. The production of these vectors in sufficient quantities for clinical applications remains a hurdle, prompting the field toward developing suspension processes that are conducive to large-scale production. Advanced upstream bioprocessing approaches will need to be complemented by appropriate downstream processes in order to reduce overall manufacturing costs and address the current viral vector supply gap. In this study, stable HEK293 producer cell lines were employed that grow in suspension, thus offering direct scalability, and producing a green fluorescent protein (GFP)-expressing lentiviral vector in the 106-7 transduction units (TU)/mL range in batch culture without optimization. HEK293 stable producer cells were retained in 3 L bioreactors operated in perfusion mode using either a BioSep acoustic cell filter (Applisens), an XCell™ ATF system (Repligen) or a VHU™ Perfusion Filter (Artemis Biosystems). Cultures were grown up to 1 – 1.5 ×106 cells/mL in batch mode. Perfusion was started at 0.5 volume of medium per reactor volume per day (VVD) and induction was carried out after reaching the targeted cell density of 5 ×106 cells/mL. Perfusion was then continued at 1 VVD with fresh medium containing inducers for 3 – 4 days. In all perfusion runs, harvests were collected and the LV-containing supernatant was kept on ice or at 4ºC until clarification (once daily) and subsequently stored at -80ºC until quantification using the GTA assay. We are currently working on bioprocess development integrating this upstream process with suitable downstream approaches supported through the use of process development-enabling analytical methods. Our study demonstrates that LV production in perfusion mode using the VHU filter outperformed our routine perfusion approach using an acoustic cell filter. Cells were retained in the bioreactor while LV particles passed through the filtration device with the harvest. Using this novel device, the cumulative functional LV titers were increased by up to 30-fold compared to batch mode, reaching a cumulative total yield of \u3e2 ×1011 TU/L of bioreactor culture. This approach is easily amenable to large scale production and commercial manufacturing. Purification processes used to manufacture LVs need to be tailored to the unstable nature of LVs to counter vector instability and yields need to be improved through process optimization, such as the application of novel purification methodologies in continuous or semi-continuous mode. We will describe what DSP strategy we will use to most effectively integrate up- and downstream processing for lentiviral vectors. We also expect that our bioprocessing strategy will be transferable to other modalities having similar properties than LV

    Accelerated mass production of influenza virus seed stocks in HEK-293 suspension cell cultures by reverse genetics

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    Despite major advances in developing capacities and alternative technologies to egg-based production of influenza vaccines, responsiveness to an influenza pandemic threat is limited by the time it takes to generate a Candidate Viral Vaccine (CVV) as reported by the 2015 WHO Informal Consultation report titled “Influenza Vaccine Response during the Start of a Pandemic”. In previous work, we have shown that HEK-293 cell culture in suspension and serum free medium is an efficient production platform for cell culture manufacturing of influenza candidate vaccines. This report, took advantage of, recombinant DNA technology using Reverse Genetics of influenza A/Puerto Rico/8/34 H1N1 strain, and advances in the large-scale transfection of suspension cultured HEK-293 cells. Transfection in shake flasks was performed using 1ug of total plasmid and 1x106 cells/mL. The supernatant was harvested after 48 hpt and used to infect a new shake flasks at 1x106 cells/mL for virus amplification. 3-L bioreactor was inoculated and transfected at 1x106 cells/mL with 1ug of total plasmid and harvested after 48hpt and the virus generated was amplified in shake flask. Quantification by TCID50, SRID, Dot-blot and TRPS were performed as well as characterization by TEM and HA and NA sequencing. Small-scale transfection in shake flasks generated 1.5x105 IVP/mL after 48 hpt and 1x107 IVP/mL after 96 hpi. For large-scale experiment a 3-L controlled stirred tank bioreactor resulted in supernatant (P0) virus titer of 5x104 IVP/mL and 2.8x107 IVP/mL after only one amplification (P1) in HEK-293 suspension cells. We demonstrate the efficent generation of H1N1 with the PR8 backbone reassortant under controlled bioreactor conditions in two sequential steps (transfection/rescue and infection/production). This approach could deliver a CVV for influenza vaccine manufacturing within two-weeks, starting from HA and NA pandemic sequences. Thus, this innovative approach is better suited to rationally design and mass produce the CVV within timelines dictated by pandemic situations and produce effective responsiveness than previous methodolog

    Pan-HA antibodies confer protection in mice against influenza

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    The elderly population is one of the most vulnerable groups to influenza infection and influenza-related complications. Unfortunately, vaccination exhibits reduced efficacy in this population. Immunization and treatment with passive antibody transfer could therefore be a valuable alternative. Please click Additional Files below to see the full abstract

    Bioengineering of a TAT-conjugated Peptide to Modulate the Activity of Glycogen Synthase Kinase-3 in Adult and Embryonic Stem Cells

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    The intracellular delivery of molecules to modulate signaling pathways and gene expression is a powerful approach to control stem cell fate decision. For applications in gene therapy and regenerative medicine, the use of genetic material and viral vectors raise concerns because stem cells persist throughout life, and long-term effects of uncontrolled genetic modifications could affect the cellular progeny. An alternative is to deliver directly peptides or proteins using cell-permeable peptides (CPPs) which have the ability of crossing the plasma membrane and carrying cargos into cells. CPPs can therefore be used to deliver factors to direct stem cells proliferation, survival and differentiation. This thesis describes an approach to control stem cell fate based on the delivery of a CPP-conjugated bioactive peptide. A first significant contribution from this work is the development of a flow cytometric assay to accurately quantify the uptake of a panel of CPPs. This study revealed that HIV-transactivator of transcription (TAT) and Antennapedia (Antp) offered the highest level of translocation in different cell types. The uptake was improved by treating the cells with a single, low-voltage electrical pulse that selectively enhances the amount of TAT-conjugated peptides and proteins delivered by at least an order of magnitude, without causing cellular toxicity or apoptosis. Subsequently, flow cytometry, confocal microscopy, capillary electrophoresis and mass spectrometry were used to examine the intracellular fate of TAT-conjugated peptides in order to define the parameters that limit their bioactivity and point to specific sequence modifications that can improve their efficacy. The advances described in this thesis were applied to the development of TAT-eIF2B, a peptide-inhibitor of glycogen synthase kinase-3 (GSK-3). TAT-eIF2B was found to be specific for GSK-3 and had a significant positive effect on the formation of neurospheres in embryonic stem cell cultures and on the survival of myeloid progenitors in cytokine-starved fetal liver cell cultures. On the other hand, GSK-3 inhibition reduced the number of neurospheres generated by human olfactory neuroepithelium cells due to lower proliferation and increased neuronal differentiation. In summary, this work describes the development of a peptide-based technology to deliver bioactive cargoes in cells, and it demonstrates its utility for modulating the activity of a master regulator of stem cell fate decision.Ph
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